Characterization of the Phenol Monooxygenase Gene from Chromobacterium violaceum: Potential Use for Phenol Biodegradation

다국어 초록 (Multilingual Abstract)

In this work, the biodegradation mechanism of phenol and sub products (such as catechol and hydroquinone) in Chromobacterium coli
was investigated by cloning and molecular characterization of a phenol monooxygenase
gene in Escherichia coli. This gene (Cvmp) is very similar (74 and 59% of similarity and identity, respectively) to the
ortholog from Ralstomia eutropha, bacteria capable of utilizing phenol as the sole carbon source. The phenol biodegradation
ability of E.coli recombinant strains was tested by cell-growth in a minimal medium containing phenol as the
sole source of carbon and release of intermediary metabolites (catechol and hydroquinone). Interestingly, during the
growth of these strains on phenol, catechol, and hydroquinone accumulated transiently in the medium. These metabolites
were further analyzed by HPLC. These results indicated that phenol can be initially orto or para hydroxylated
to produce cathecol or hydroquinone, respectively, followed by meta-cleavage of aromatic rings. To verify this information,
the metabolites obtained from HPLC were submitted to LC/MS to confirm their chemical structure, thereby indicating
that the recombinant strains utilize two different routes simultaneously, leading to different ring-fission substrates
for the metabolism of phenol

다국어 초록 (Multilingual Abstract)

In this work, the biodegradation mechanism of phenol and sub products (such as catechol and hydroquinone) in Chromobacterium coli
was investigated by cloning and molecular characterization of a phenol monooxygenase
gene in Escherichia coli. This gene (Cvmp) is very similar (74 and 59% of similarity and identity, respectively) to the
ortholog from Ralstomia eutropha, bacteria capable of utilizing phenol as the sole carbon source. The phenol biodegradation
ability of E.coli recombinant strains was tested by cell-growth in a minimal medium containing phenol as the
sole source of carbon and release of intermediary metabolites (catechol and hydroquinone). Interestingly, during the
growth of these strains on phenol, catechol, and hydroquinone accumulated transiently in the medium. These metabolites
were further analyzed by HPLC. These results indicated that phenol can be initially orto or para hydroxylated
to produce cathecol or hydroquinone, respectively, followed by meta-cleavage of aromatic rings. To verify this information,
the metabolites obtained from HPLC were submitted to LC/MS to confirm their chemical structure, thereby indicating
that the recombinant strains utilize two different routes simultaneously, leading to different ring-fission substrates
for the metabolism of phenol

참고문헌 (Reference)

1 Watanabe, K., "Understanding the diversity in catabolic potential of microorganisms for the development of bioremediation strategies" 81 : 655-663, 2002

2 Grangeiro, T. B., "Transport genes of Chromobacterium violaceum: An overview" 31 : 117-133, 2004

3 Keener, W. K., "Transformations of aromatic compounds by Nitrosomonas europaea" 60 : 1914-1920, 1994

4 Brazilian National Genome Project Consortium, "The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability" 100 : 11660-11665, 2003

5 Neumann, G., "Simultaneous degradation of atrazine and phenol by Pseudomonas sp. strain ADP: effects of toxicity and adaptation" 70 : 1907-1912, 2004

6 Kivisaar, M., "Selection of independent plasmids determining phenol degradation in Pseudomonas putida and the cloning and expression of genes encoding phenol monooxygenase and catechol 1,2-dioxygenase" 24 : 25-36, 1990

7 Neujahr, H. Y., "Phenol hydroxylase from yeast. Purification and properties of the enzyme from Trichosporon cutaneum" 35 : 386-400, 1973

8 Hino, S., "Phenol hydroxylase cloned from Ralstonia eutropha strain E2 exhibits novel kinetic properties" 144 : 1765-1772, 1998

9 Monteiro, A. A. M. G., "Phenol biodegradation by Pseudomonas putida DSM 548 in a batch reactor" 6 : 45-49, 2000

10 Shingler, V., "Nu-cleotide sequence and functional analysis of the complete phenol/3,4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600" 174 : 711-724, 1992

11 Watanabe, K., "Molecular detection, isolation, and physiological characterization of functionally dominant phenol-degrading bacteria in activated sludge" 64 : 4396-4402, 1998

12 Thakur, I. S., "Molecular cloning and characterization of pentachlorophenol-degrading monooxygenase genes of Pseudomonas sp. from the chemostat" 290 : 770-774, 2002

13 Sambrook, J., "Molecular Cloning: A Laboratory Manual. 2nd ed" Cold Spring Harbor Laboratory Press 23-28, 1989

14 Futamata, H., "Group-specific monitoring of phenol-hydroxylase genes for a functional assessment of phenol-stimulated trichloroethylene bioremediation" 67 : 4671-4677, 2001

15 Jones, K. H., "Evidence of two pathways for the metabolism of phenol by Aspergillus fumigatus" 163 : 176-181, 1995

16 Neujahr, H. Y., "Degradation of phenols by intact cells and cell-free preparations of Trichosporon cutaneum" 13 : 37-44, 1970

17 Kukor, J. J., "Complete nucleotide sequence of tbuD the gene encoding phenol/cresol hydroxylase from Pseudomonas pickettii PKO1 and functional analysis of the encoded enzyme" 174 : 6518-6526, 1992

18 Nordlund. I., "Complete nucleotide sequence and polypeptide analysis of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600" 172 : 6826-6833, 1990

19 Duran, N., "Chromobacterium violaceum: a review of pharmacological and industrial perspectives" 27 : 201-222, 2001

20 Santos, P. M., "Characterization of the unique organization and co-regulation of a gene cluster required for phenol and benzene catabolism in Pseudomonas sp. M1" 131 : 371-378, 2007

21 Chen, W. M., "Characterization of phenol and TCE degradation by the rhizobium Ralstonia taiwanensis" 155 : 672-680, 2004

22 Takeo, M., "Characterization of alkylphenol degradation gene cluster in Pseudomonas putida MT4 and evidence of oxidation of alkylphenol and alkylcatechols with medium-lenght alkyl chain" 102 : 352-361, 2006

23 Subramanyam, R., "Biodegradation of catechol (2-hydroxy phenol) bearing wastewater in an UASB reactor" 69 : 816-824, 2007

24 Heinaru, E., "Biodegradation efficiency of functionally important populations selected for bioaugmentation in phenol- and oil-polluted area" 51 : 363-373, 2005

25 Arai, H., "Adaptation of Comamonas testosteroni TA441 to utilize phenol: organization and regulation of the genes involved in phenol degradation" 144 : 2895-2903, 1998

26 Chen, W. P., "A simple and rapid method for the preparation of gram-negative bacterial genomic DNA" 21 : 2260-, 1993